A range of different polyphenols can be ingested in a bowl of polyphenol-rich fruit, going from one-phenol hydroxybenzoic acid to more complex polymeric compounds. Epidemiologically, polyphenol consumption has been associated with a reduced risk of cancer and cardiovascular disease and neurological protection against brain ageing. However, only a small proportion of native polyphenols (5-10%) are absorbed and the remainder reach the colon, where they are extensively metabolised by the gut microbiota. The colonic microbiota produces a relatively small number of polyphenol microbial metabolites from a large number of different dietary polyphenols. During subsequent tissue distribution, the target organs and the effective concentration circulating remain largely unreported. This Ph.D. thesis is divided into two parts: chemical analysis of food composition and in vivo bioavailability of polyphenol metabolites. Metabolomics offers an innovative approach that has recently been shown to be effective in both food chemistry and nutritional bioavailability studies. Polyphenol composition in strawberries is studied in the first part of this Ph.D. thesis, with the aim of evaluating nutritionally significant amounts of polyphenols before ingestion. In this context, a targeted method for quantitative analysis of multiple classes of phenols was developed. A high sensitivity MRM-based method for 135 phenolics with a wide dynamic range was obtained, providing valuable insight and assisting with the analysis of complex matrices such as fruit, and more in general food. Application of the method was tested in Fragaria spp., and along with another rapid method for the analysis of anthocyanins and ellagitannins, provided a general overview of polyphenol composition in strawberries. A total of 56 individual compounds were accurately identified and quantified, some of them for the first time, their concentration ranging from 1 ug/100 g for low abundant polyphenols to 40 mg/100 g of fresh fruit. Moreover the isolation of some ellagitannins and definition of their profile in Fragaria spp. was carried out during fruit ripening. Clarification of the main ellagitannin, agrimoniin, was obtained by isolation and it was ambiguously assigned as the main ellagitannin present in the diet. In the second part of this Ph.D. thesis the in vivo bioavailability of a dose of polyphenol microbial metabolites reflecting dietary consumption of fruit was studied. The focus was on the metabolites of polyphenols which can be found in the bloodstream after gut microflora metabolism. Their distribution was explored in rats in different organs, in particular in the brain, considering their possible neuropreventive properties. Development of a specific quantitative method for the quantification of selected polyphenol microbial metabolites made it possible to analyse complex biological samples resulting from in vivo trials with rats treated with a nutritionally significant dose of polyphenol microbial metabolites, intravenously injected. A high-throughput, sensitive and reproducible method for 23 polyphenol metabolites in six different biological matrices was developed. A purification protocol made it possible to obtain cleaner and more concentrated samples, with low limits of quantification. Specific organ-tropism was observed, mainly hepatotropism. Remarkably, in this study the brain was reported to be one of the target organs for these molecules, already being present at basal level or increasing their concentration after treatment. Furthermore, the amount of 10 out of 23 compounds significantly increased with a nutritionally significant dose.

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.

Glioblastoma multiforme (GBM) is the most common and aggressive primary brain tumor in adults. The search for new effective chemotherapeutic agents to treat GBM has proven challenging throughout the last few decades. As a result, very limited pharmacological treatment is currently available. GBM aggressiveness is associated with its glioblastoma stem cells (GSCs) component, which is responsible for resistance to therapy. Therefore, new specific pharmacological approaches directed to eradicate GSCs are endowed with a great therapeutic potential. GSCs have been shown to rely on mitochondrial respiration for their high energy demand. In order to have a functional mitochondrial respiration process, the five complexes forming the oxidative phosphorylation (OXPHOS) chain have to be built by the coordinate assembly of proteins translated by either the cytosolic or the mitochondrial ribosomes. Given their endosymbiotic origin and despite the evolutionary changes occurred the mitochondrial ribosomes (mitoribosomes) still share structural and functional similarities with the bacterial ones, particularly considering the functional ribosomal core. In the light of these similarities, we hypothesized that antibiotics targeting bacterial ribosomes could be exploited to inhibit mitoribosomes, affecting mitochondrial translation and OXPHOS assembly, and hence leading to detrimental effect on GSCs viability. We performed a high-content imaging driven screening of several bacterial ribosome targeting antibiotics and identified Drug A as the most promising compound due to its cytotoxic and mitotoxic effects on GSCs. We demonstrated that Drug A effectively prevents GSCs expansion, resulting to be over an order of magnitude more effective in GSCs growth inhibition than temozolomide, the only drug used in first line GBM therapy. We then investigated the mechanism of action of Drug A, proving that it inhibits mitochondrial translation and, as a consequence, it decreases the functionality of the OXPHOS complexes reducing mitochondrial respiration capacity. Moreover, we obtained the structure of this compound bound to the human mitoribosome using cryo-electron microscopy, which provides the basis for further development of more potent analogs. Finally we proved the efficacy of Drug A in vivo using a xenograft mouse model of GBM. Our results suggest that mitochondrial translation represents a therapeutic target for GBM and show that Drug A, acting via inhibition of mitochondrial translation, is extremely effective against GSCs. Given the urgent medical need for novel therapeutic approaches in GBM treatment, Drug A represents a promising therapeutic solution that is worth further preclinical and clinical investigations.