In the last decade there has been a new reappraisal of the function of lipids in the cell life, not only for their structural role as cell wall or for their energy storage function, but also for their significant function on signalling and protein recognition processes. This new attention on lipids has led to a new research field in the metabolomics world called “Lipidomics”. Lipidomics is more than just the complete characterization of all lipids in a particular biological sample. It is the comprehensive understanding of the influence of all lipids on a biological system with respect to cell signalling, membrane architecture, transcriptional and translational modulation, cell-cell and cell-protein interaction and response to environmental changes over time. The critical role of lipids in cell, tissue and organ physiology is already demonstrated by many human diseases involving the disruption of lipid metabolic enzymes and pathways. Examples of such diseases include diabetes, cancer, neurodegenerative disorders and infectious diseases. This represents a clue for understanding the molecular diversity observed in membrane phospholipids. Subtle biophysical properties are also another possible explanation of strong interest toward lipids especially with reference to the emerging field of heterogeneous membrane microdomains (rafts). The major goal in lipidomics is the identification of metabolic pathways which are activated or deactivated during development of an organism or when a cell is shifted from an established physiological condition to another physiological or pathological condition (metabolic learning). A better understanding of the regulation of underlying metabolic pathways is necessary to design novel strategies for intervention. In this thesis attention has been focused on two biological systems to demonstrate how the study of membrane lipids can help for a better understanding of the mechanisms involved in different biochemical processes. The first system tackled by our methodology is represented by lipid components in detergent resistant membranes (DRM) associated with the expression of Prostate-specific membrane antigen (PSMA); the latter is a 750-residue type II transmembrane glycoprotein of the normal prostate cells and one of the most promising biomarkers of prostate carcinoma as its expression is drastically increased in cancer cells protein involved in the prostate carcinoma. In particular, the complex glycosylated form of the protein is found in Lubrol insoluble DRMs. Many essential cellular events, such as protein sorting, endocytosis and signal transduction pathways, are triggered via association of the proteins directly implicated in these processes with DRMs. In the present work we report on the lipid composition of PSMA-associated microdomains. A qualitative screening was made by thin layer chromatography (TLC) followed by an extended NMR analysis of the sample. In particular, by taking 1H-, 13C- ed 31P-NMR spectra we were able to detect and quantify cholesterol and the relative contribution of all the lipids belonging to a given PL class (such as PC, PE and SM) with respect to the overall PL composition. On the other hand, Electrospray Ionization (ESI-MS) measurements and in-source collisional induced dissociations (CID) carried out on the same sample allowed us not only to establish the distribution of lipids among the PL classes but also to recognize some structural features such as the length and the number unsaturations of their acyl chains. The second problem tackled by following similar analytical methodologies was to analyze the detailed membrane lipids profile of insect larvae of the species Pseudodiamesa branickii and Diamesa cinerella to obtain information about the biochemical mechanisms involved in their thermal adaptative capabilities. Through NMR and LC-ESI-MS experiments we have obtained information about the composition of the lipid pool of these larvae that is formed essentially by triglycerides and phospholipids, with a variable inter-species PC/PE ratio. Coupling LC-MS profiling methods with in-source fragmentation data further has enhanced the ability to probe the lipidome, by supplementing lipid identification with structural information. We have observed an high level of unsaturation of the fatty acid chain as expected for low temperature-adapted species since unsaturated chain promote a more disordered and flexible membrane structure.. For Diamesa cinerella, from ESI data we could also infer some possible mechanism adopted to increase unsaturation of the fatty acid chain by the action of desaturases enzymes or by the insertion of PUFA chains.

Lipids were once thought to only be the building blocks of cell membranes and to serve as energy reserves. With time however, it became increasingly clear that they are actually involved in many more roles. Not surprisingly, the comprehensive characterisation of lipids in cells and tissues has experienced a growing interest worldwide, to the point that the term "lipidomics" was coined. This field is a subset of metabolomics, and the interesting point about these two sciences is that they are closest to the phenotype as compared to their "omics" counterparts (genomics, trascriptomics, ...), because metabolites and lipids are the end products of the –omics cascade. We have investigated mass spectrometry-based lipidomics from different perspectives: first of all, we have devised a targeted approach in which we have focused on sphingolipids and their perturbations. We started by working on neuronal cell cultures where we inhibited GBA, a key enzyme of the sphingolipid metabolism known to be one of the risk factors for Parkinson's disease. We found a significant sphingolipid unbalance characterised by an accumulation of glycosyl-ceramides. We then moved on by investigating the effects that LRRK2, an important and complex protein known to be related to autosomal-dominant forms of the disease, has on sphingolipids. We worked on mouse models, and we compared the sphingolipid profiles of wild-type (Lrrk2+/+) and knock-out (Lrrk2–/–) mice, finding a marked increase in ceramide levels and, more in general, in all lipids downstream of GBA. Such results hint to a possible interaction between LRRK2 and GBA, with LRRK2 playing a role in GBA regulation. In a second lipidomics investigation, we tried to understand whether or not anti-cancer treatments affect the lipid composition of tumours. Specifically, we concentrated on a common anti-angiogenic drug, whose aim is to starve cancer cells by inhibiting angiogenesis, a process required by the tumours to grow. We considered four different adenocarcinoma cell lines, which were subcutaneously inoculated into mice; the "control" animals received no treatment, whereas the "treated" ones were periodically given the drug. Interestingly, we found the treatment to have significant effects on the cancer lipidome, although the different lines responded unequally to the drug. Such results may reflect the huge heterogeneity of cancers and of individual responses to the treatment. Finally, we developed an informatics algorithm that deals with labelling experiments. The key point is that mass spectrometry measures isotopic patterns of analytes, which depend on the isotopic distribution of the elements; consequently, if an analyte incorporates the stable isotope employed in a labelling experiment, it will show a modified isotopic pattern. Our algorithm analyses such pattern, estimating the abundance of the incorporated label; we first tested it over carefully planned samples, and then we used it in a biochemical application where we wished to establish whether the rate of de novo lipogenesis is influenced by diet. This was accomplished by designing an experiment where mice were given partially deuterated water, while being fed different diets; we were able to ascertain that diet does indeed affect de novo lipogenesis, with the lowest rates occurring on fat-rich diets. We are confident that our tool may find useful applications, considering that stable isotope-based labelling experiments are becoming more and more popular.

Acquired Immunodeficiency Syndrome (AIDS) is a disease caused by the Human Immunodeficiency Virus (HIV). Since its discovery in 1981, more than 25 million people died due to this disease. To date, an effective HIV-1 vaccine usable in prophylaxis or in the therapy of humans has not yet been identified. The failures and limited success of HIV vaccines have reinforced the role of chemotherapy and therefore research on the development of effective drugs. Non-nucleoside reverse transcriptase inhibitors (NNRTIs) were the early agents introduced in the therapy and currently they are the most used, based on their concurrent high activity against the virus and low toxicity against human cells. In addition, the rapid development of virus resistance against these types of drugs, needs to find new molecules able to overcome this drawback. My thesis work started from the design of a small library of new molecules, with hybrid structures based on a template deriving from the natural product (+)-calanolide A and the synthetic molecule α-APA, both showing a potent and selective activity against reverse transcriptase. Docking calculation has allowed to select molecules having the best values of interaction energy with the viral enzyme. Chemical synthesis was carried out together with structural characterization by extensive spectroscopic analysis including NMR technique and mass spectrometry. In particular, the synthesis of the amide group present in the structure of some amino-pyrone compounds using the standard method, resulted in the expected N-acylation, but with a C-acyl byproduct. This result has suggested to look further into the study of N,C-acylation selectivity for the ambidentate amino-pyrone moiety, whose reactivity is poorly known. Regioselectivity was investigated under different conditions (organic bases, solvent, acylating agent), also for an enamino-ester taken as a model compound. Experimental procedures were optimized in order to synthesize selectively pure N- and C-acylated compounds. A preliminary enzymatic assay indicated a good activity in the early prepared compounds of the series, promising for the following in vitro tests on HIV infected cells of each molecule in the whole series. In addition, these compounds were tested against other common viruses for human infective pathologies. With the aim of identifying molecules with potential therapeutic applications, the antiviral activity must be related to cytostatic effect, in order to select the ones with a favored selectivity index. Unfortunately, the molecules showed paragonable values in antiviral and cytostatic effects, the latter one not easily predictable neither by the chemical structure, nor by a computational approach. If the drug design by molecular docking has failed in selecting a new scaffold for NNRTIs, the study has driven the interest towards new potential antitumoral molecules showing activity at sub-micromolar concentration against leukemic cell lines. Due to the structural similarity with recently studied antibacterial natural pyrones, the synthetic molecules showing the lowest values of cytotoxicity were investigated in the inhibition of bacterial strains. Some tested compounds have shown a good activity and selectivity against Gram(+) bacteria.

Resveratrol is a polyphenolic compound produced by various plants and present in dietary sources such as red wine. In recent years, its beneficial effects for human health, including protection from heart diseases and cancer prevention, have attracted increasing interest. Resveratrol acts both as an antioxidant and a prooxidant agent when works in vivo with Cu(II) ions occurring naturally in living organisms. The aim of this work is to study the gas phase reactivity of resveratrol in presence of copper and iron ions, in order to more insights on the role of copper in the proposed biological mechanism. By electrospray ionization (ESI) mass spectrometry we have produced and detected some resveratrol-copper complexes by using a resveratrol/CuSO4 solution in acetonitrile/water, and their most stable structures have been calculated at the B3LYP/6-311G(d) level of theory. The formation of dehydrodimer product was also detected in ESI-MS/MS experiments and its structure assigned with evidences for isomeric compounds from copper and iron reactions with resveratrol. Density Functional Theory (DFT) calculations have been carried out to elucidate reaction mechanisms. Finally, the crucial role of the para-OH group in resveratrol structure has been demonstrated by investigating reactions with copper sulfate of synthetic analogues, bearing different number and position of OH groups.

Iron-sulfur clusters are indispensable to extant metabolism and are thought to have had an ancient role in mediating the chemical reactions that led to life. However, there has been no clear proposal for how these inorganic clusters came to occupy such an important position in biology. In this thesis I describe my efforts in delineating a plausible path from short, prebiotically plausible peptides to longer sequences with similar features to modern day iron-sulfur proteins. Small organic thiolates and short cysteine-containing peptides can give rise to [2Fe-2S] and [4Fe-4S] clusters in aqueous solution when irradiated with UV light in the presence of iron ions. Additionally, duplications of tripeptides coordinated iron-sulfur clusters give sequences which are better able to stabilize iron-sulfur clusters, resembling motifs with cysteinyl ligand spacing highly similar to contemporary ferredoxins. Moreover, the studied iron-sulfur clusters are redox active and are able to mimic extant metabolic pathways, such as the first step of the electron transport chain, within protocells favouring the formation of a proton gradient which could be exploited for central biosynthetic processes.