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Investigation of Biological Membranes by NMR and ESI-MS MethodologiesSandron, Tommaso January 2010 (has links)
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.
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Design, Synthesis and Characterization of new Supramolecular ArchitecturesBaroncini, Massimo <1979> 03 June 2010 (has links)
No description available.
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New Analytical Methodologies at the Frontier of Cellular LipidomicsFerrazza, Ruggero January 2017 (has links)
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.
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Design, Chemical Synthesis and Biological Evaluation of Potential New Antiviral AgentsDefant, Andrea January 2012 (has links)
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.
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An Electrospray Ionization Mass Spectrometric Study on Reactivity of Resveratrol Induced by Metal IonsTamboli, Vajir January 2011 (has links)
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.
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Prebiotic Synthesis of Redox-Active Iron-Sulfur ClustersBonfio, Claudia January 2017 (has links)
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.
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Seleno-diamminoacidi Redox con attività GPx nello studio del danno ossidativo indotto da Aflatossine / Redox, GPx mimic seleno-diamino acids in the study of oxidative damage induced by aflatoxins.NUCCI, ADA 23 February 2012 (has links)
La contaminazione da micotossine può coinvolgere tutti i comparti della filiera agroalimentare e rappresenta a tutti gli effetti un potenziale di perdita economica.
Il settore zootecnico è particolarmente esposto alle ricadute della contaminazione, in termini di costi sanitari per gli effetti cronici sulla salute animale conseguenti all’esposizione alle micotossine.
La prevenzione in campo mediata da appropriate tecniche agronomiche rappresenta, per quanto ovvio, la più importante strategia per ridurre la contaminazione dei mangimi. Un approccio completamente differente è quello che si basa sulla possibilità di ridurre gli effetti delle tossine sull’animale, modificando la sua alimentazione attraverso l’integrazione nutrizionale di opportuni agenti chemiopreventivi.
Numerose sono le specie chimiche, di origine naturale o sintetica, che hanno dimostrato una efficacia chemiopreventiva del danno ossidativo indotto da micotossine ed emergono per la loro efficacia il selenio e i suoi composti organici ed inorganici.
L’azione antineoplastica del selenio è nota da molto tempo e ha trovato conferme in numerosi studi epidemiologici che indicano una relazione inversa tra assunzione di selenio con la dieta e rischio di sviluppo di una patologia neoplastica.
Questa tesi di dottorato ha avuto come obiettivo la progettazione, la sintesi e lo studio di composti diamminoacidici contenenti un atomo di calcogeno (selenio o zolfo), mimici dalla glutatione perossidasi (GPx) e auspicabilmente attivi nel contrasto del danno ossidativo indotto dall’aflatossina B1 (AFB1).
Le specie diamminoacidiche sintetizzate appartengono alla classe dei Sec-derivati, ovvero sono costitute da un residuo di L-selenocisteina (o L-cisteina) a cui è legato attraverso l’atomo di calcogeno un altro L-amminoacido. Alla porzione calcogenica, che rappresenta il sito redox, sono stati infatti ancorati rispettivamente un residuo di L-prolina modificata e un residuo di L-leucina.
Al termine delle fasi di sintesi e caratterizzazione chimico-fisica dei calco-diamminoacidi è stata intrapresa una serie di indagini biochimiche in collaborazione con due distinti laboratori in Germania. I risultati ottenuti hanno permesso di dimostrare che alcuni Sec-derivati sono in grado di ridurre i perossidi attraverso un meccanismo catalitico che mima l’attività della GPx. Essi sembrano inoltre evidenziare che il “meccanismo antiossidante” sia mediato sostanzialmente dall’attività GPx mentre non sono particolarmente attivi meccanismi di radical scavenging né di puro trasferimento elettronico.
Alcuni dei Sec-derivati sintetizzati sono stati oggetto di un’indagine cellulare preliminare volta a verificare una eventuale effettiva azione protettiva dei calco-diamminoacidi verso il danno cellulare indotto da AFB1. Le indagini sono state effettuate adoperando una linea cellulare HepG2.
I dati ottenuti, nel loro insieme, hanno mostrato che i calco-diamminoacidi esaminati sono di per sé caratterizzati da una notevole attività biologica. In particolare, alla concentrazione più elevata utilizzata (100 µM), alcuni di essi hanno mostrato interessanti proprietà citoprotettive contro il danno indotto da AFB1. L’efficacia è paragonabile, e in qualche caso superiore, a quella riscontrabile per il composto di riferimento, la Se-metil-selenocisteina, principale forma di selenio organico che è naturalmente presente in specie vegetali quali Allium Sativum e Brassica juncea. / Mycotoxin contamination may involve all fields of the food agricultural chain and might potentially determine economic losses. In particular, the livestock sector is at risk of health costs, as a consequence of chronic diseases induced by mycotoxin exposure.
It is obvious, although actual, that the mycotoxin contamination should be controlled on the field by a series of suitable agriculture practices in order to minimize the mycotoxin formation in feeds.
Recently, dietary strategies (antioxidant compounds, medicinal herbs, plant extracts) to counteract the effects of mycotoxins have attracted considerable attention.
Chemoprevention refers to the use of naturally occuring and/or synthetic chemicals to inhibit the development of mycotoxin induced chronic diseases. Selenium and its organic and inorganic compounds, have emerged as outstanding chemoprevention agents.
Selenium antineoplastic properties have been well documented through the last decades, being supported by several epidemiological evidences on the association between selenium intake and the risk for some kinds of human/animal cancers.
This PhD thesis was focussed on design, synthesis and study of a family of chalcogenic diamino acids (containing either selenium or sulfur), GPx mimics and potentially active to reduce the oxidative damage induced by aflatoxin B1 (AFB1).
The diamino acids synthesized belong to the family of Sec-derivatives and consist of L-selenocysteine (or L-cysteine) linked through the calchogen atom to either a L-proline or a L-leucine moiety.
The GPx-like catalytic activity of the various compounds was confirmed using both thiophenol and GPx assays. Their possible radical-scavenging activity, and electron-donor reducing character as well, could be ruled out by suitable assays.
The cytoprotective capacity exhibited by several of the compounds synthesized was investigated in HepG2 cells by MTS assays. The results indicated that all the chalcogenic diamino acids tested had a strong biological activity.
In particular, the pre-treatment of the test cells with two Sec-derivatives, employed at higher doses (100 µM), led to a protective effect on cell toxicity, lowering the AFB1-induced cell mortality, and this efficacy is comparable with that of the reference Se-metil-selenocysteine, main source of organic selenium in plants such as Allium Sativum and Brassica juncea.
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